When blood vessels are injured, blood escapes from the vascular space and coagulates. The injured blood vessels are closed by the coagulating blood and, in that manner, protect the organism from massive blood loss. Blood coagulation is caused by the enzyme thrombin, which is generated from its zymogen, prothrombin, and causes the transformation of the protein fibrinogen, which is present in blood plasma, into insoluble fibrin. The exiting blood coagulates within minutes, with only one sixth of the fibrinogen contained in plasma being transformed into fibrin. Thrombin generation continues in the coagulated blood, which still contains large amounts of fibrinogen, until all of the residual fibrinogen in the coagulated blood has been transformed into fibrin. This process of continued thrombin formation and coagulation proceeds slowly over a prolonged period of time and may take several hours until it is completed.
When fibrinogen is transformed into fibrin, thrombin causes fibrinopeptides A and B to split or partially split from the two ends of the fibrinogen molecule, whereby fibrin monomers are formed, which remain soluble for some time. These aggregate laterally to fibrils, forming a fibrin network in the further course (Blombäck). This fibrin network, which has formed in the coagulated blood, adheres to the injured tissue or wound bed and, in that manner, leads to hemostasis and wound closure.
Thrombin also causes the transformation of coagulation factor XIII, the zymogen of coagulation factor XIIIa, into a transglutaminase. The latter cross-links proteins such as fibrin, fibrin monomers, fibrinogen, and other proteins occurring in blood plasma by covalent bonds. The cross-linking of fibrin, in particular, is of great importance for the stability of the nascent fibrin network (Siebenlist et al.).
Thrombin also transforms the thrombin-activatable fibrinolysis inhibitor (TAFI) into a carboxypeptidase (TAFIa), which splits carboxyterminal lysine residues from fibrin and thereby inhibits the formation of the tissue-plasminogen-activator-plasminogen-fibrin-complex, which is necessary for the transformation of plasminogen into plasmin (Booth).
In vitro, the formation of thrombin in blood or blood plasma and thus the onset of the coagulation process is rendered possible by tissue extracts. Such extracts can best be obtained from brain using aqueous media or organic solvents (Morawitz).
The coagulation-active material, which can be extracted using a suitable buffer and which is termed thromboplastin, consists of an apoprotein, the tissue factor, and coagulation-active lipids. Small amounts of thromboplastin, when added to blood or plasma, suffice to generate rapid coagulation.
Using organic solvents, it is possible to separate the coagulation-active lipids from apoprotein. The aqueous apoprotein part is then termed partial thromboplastin, which, in the presence of kaolin, glass powder, and other surface-active substances also leads to rapid coagulation when blood or blood plasma are added.
This has led to the concept of an activation cascade of enzymes resulting in the formation of the enzyme thrombin, which causes blood coagulation (Davie et al.; MacFarlane).
The coagulation process which is triggered by thromboplastin is referred to as the extrinsic pathway of blood coagulation, in contrast to the coagulation process which is triggered by partial thromboplastin, the latter being termed the intrinsic pathway. The two processes have in common that they transform coagulation factor X into Xa, albeit by different routes. Accordingly, a differentiation is made between the extrinsic and intrinsic tenase pathways in the first part of the coagulation process, which leads to activation of coagulation factor X. In another enzyme complex, prothrombinase, coagulation factor Xa converts prothrombin into thrombin. This process is referred to as the common pathway. The enzyme system which generates coagulation factor Xa with the aid of thromboplastin is termed the extrinsic tenase complex, in contrast to the enzyme system in which partial thromboplastin plays a role, i.e. the intrinsic tenase complex.
The prevailing view has it that tissue factor, jointly with coagulation factor VIIa and thrombocytes (i.e., platelets) trigger blood coagulation after injuries (Rapaport et al.). Tissue factor occurs in almost all tissues in highly variable amounts along with coagulation-active lipids, and the two substances, when in contact with blood, form the extrinsic tenase complex, since small amounts of activated coagulation factor VII are always present in blood (Drake et al.). The extrinsic tenase complex transforms both, coagulation factors X and IX into Xa and IXa, respectively, and IXa transforms X into Xa. The activation of factor X and the resulting formation of thrombin, however, come to a halt rapidly by the tissue factor pathway inhibitor (TFPI). The temporarily formed extrinsic tenase complex as well as coagulation factor XIa independently lead to the activation of the intrinsic tenase complex, which, as far as the activation of coagulation factor X is concerned, is 50-fold more active than the extrinsic tenase complex. The intrinsic tenase complex consists of activated coagulation factors IXa, VIIIa, and coagulation-active phospholipids and is not inhibited by TFPI (von dem Borne et al.). In the intrinsic pathway, the activity of the enzyme coagulation factor IXa, which transforms coagulation factor X in Xa, is increased 100,000 to 1,000,000-fold. This increase is caused by co-factor VIIIa, which itself is not an enzyme, and certain phospholipids at an optimum calcium ion concentration.
Since the intrinsic tenase complex transforms coagulation factor X into Xa very rapidly, and the latter, jointly with cofactor Va and coagulation-active phospholipids, activates prothrombin, great amounts of thrombin are formed in a very short time. (Mann et al.).
The individual coagulation factors and platelets—the essential components of blood responsible for the process of coagulation—are normally present in abundance. Only if one of these components is reduced by 90% or more, an increased propensity to bleeding can be noticed. Bleedings become life-threatening only in deficiency states where a coagulation factor and/or platelets drop to several percent of their normal values. The central importance of tissue factor in triggering the coagulation process and its dissemination in all organs is beyond doubt, however, severe disturbances of blood coagulation occur in tissues with low tissue factor content in cases where a factor of the intrinsic tenase complex or of the prothrombinase complex is pathologically reduced. A case in point are patients suffering from hemophilia A or B. The blood of these patients still coagulates in most instances, however, because the intrinsic tenase complex is deficient or absent, thrombin formation in the coagulated blood is insufficient and the clot dissolves rapidly, so that no satisfactory hemostasis is achieved.
Thrombin, mostly of bovine origin, is used as a medicinal product for non-parenteral administration to achieve hemostasis in cases of superficial injuries. Its hemostyptic effect could be improved decisively when administered jointly with medicinal products containing fibrinogen (Grey; Young et al.). Because of their species-specific use, fibrinogen and thrombin are obtained primarily from allogenic source material today. By combining the application of thrombin with fibrinogen-containing medicinal products, one attempts to mimick and improve the physiological blood coagulation and accompanying hemostasis. This can be achieved also in patients with severe blood coagulation disturbances (Matras et al.).
By combining the application of fibrinogen concentrates, whose fibrinogen content amounts to 10- to 20-fold the fibrinogen content of blood, and great amounts of thrombin (100-1000 Upper mL), it is possible to reduce the coagulation time in such a fibrinogen-thrombin mixture to a matter of seconds and obtain a 10 to 100-fold reduction compared to the physiological bleeding time. This has made it possible to practically achieve instantaneous hemostasis when such fibrinogen-thrombin mixtures are applied in an optimal manner, provided that no larger blood vessels, particularly arterial vessels, were injured (Spängler).
The fibrinogen transformed into fibrin by thrombin adheres to the wound bed as does coagulated blood, the transglutaminases which were activated by the action of thrombin obviously causing covalent bonds between the injured tissue and the fibrin formed. This strong adherence of the formed fibrin to tissue can be used also to glue non-bleeding tissue, since the formed fibrin does not impair the healing of the glued tissue in most cases and is largely degraded in a matter of days or weeks (Matras et al.).
When fibrinogen-thrombin mixtures are used to achieve hemostasis, an as rapid as possible initiation of the coagulation process is desirable. In contrast, a gradual onset of the coagulation process is preferable in cases where parts of tissue are glued and also for sealing purposes. This makes it possible for the tissue parts to be adapted more appropriately, and similar adaptations are necessary in sealing. Thus far, a slowing of the coagulation process has been achieved by a reduction of the thrombin concentration to approximately 1% of the amount of thrombin used to achieve hemostasis. However, the use of both, high and low concentrations of thrombin is accompanied by disadvantages.
Highly viscous fibrinogen solutions containing between 5 and 10% fibrinogen can be brought to coagulate with thrombin amounts ranging from 100 to 1000 units within seconds. This short coagulation time is necessary to achieve hemostasis rapidly after the application of such a mixture to a bleeding site and arrest the bleeding. The disadvantage of such a procedure is that the fibrinogen and thrombin solutions are poorly mixed, since the high viscosity of the mixture does not allow satisfactory mixing in a short time. What results is a non-homogenous coagulation with an attendant impairment in biomechanical quality.
On the other hand, if fibrinogen-thrombin mixtures are used not to achieve hemostasis but rather to glue parts of tissue, it is necessary in most cases to slow down the coagulation of the fibrinogen-thrombin mixture in order to be able to optimize the adaptation of the parts to be glued or sealed before coagulation occurs. At present, this is attempted by reducing the amount of thrombin to between one tenth to one hundredth of the amount of thrombin used to achieve hemostasis at the disadvantage that not all of the fibrinogen present is converted into fibrin and not all of the factor XIII is converted to factor XIIIa. This procedure is also not suitable for obtaining an optimal fibrin clot, since the low thrombin concentration is not sufficient to transform TAFI into TAFIa.
A further problem is the use of bovine materials for the manufacture of medicinal products containing thrombin. Since a risk of transmission of prions by any bovine organs cannot be excluded with certainty, bovine thrombin is hardly used anymore today.
Bovine thrombin has the further disadvantage that it is antigenic for other species and can provoke allergies and anaphylaxes. In addition, patients treated with bovine thrombins have been observed to develop coagulation disorders, which is attributable to the fact that bovine thrombin can cause the formation of antibodies against coagulation factors which cross-react with human coagulation factors, thus retarding the coagulation process.
In manufacturing thrombin from prothrombin, thromboplastin from animal source material, mostly bovine brain, has often been used to activate thrombin, which increases the yield. Because of the risk of transmission of prions, thromboplastin from bovine source is rarely used anymore in the manufacture of thrombin, and a poor thrombin yield is put up with instead.